The technology for the functional expression of DNA fragments in heterologic genetic systems depends to a great extent on an accessible source of DNA. There are two ways to obtain genetic material for genetic engineering manipulations: (1) isolation and purification of DNA in an appropriate form from natural sources (this technique is well-elaborated and constitutes the backbone of genetic engineering and molecular biology), or (2) the synthesis of DNA using various chemical-enzymatic approaches, a discipline that has been intensively researched over the last 15 years. The former approach is limited to naturally-occurring sequences which do not easily lend themselves to specific modification. The latter approach is much more complicated and labor-intensive. However, the chemical-enzymatic approach has many attractive features including the possibility of preparing, without any significant limitations, any desirable DNA sequence.
Two general methods currently exist for the synthetic assembly of oligonucleotides into long DNA fragments. First, oligonucleotides covering the entire sequence to be synthesized are first allowed to anneal, and then the nicks are repaired with DNA ligase. The fragment is then cloned directly, or cloned after amplification by the polymerase chain reaction (PCR). The DNA is subsequently used for in vitro assembly into longer sequences. This approach is very sensitive to the secondary structure of oligonucleotides, which interferes with the synthesis. Therefore, the approach has low efficiency and is not reliable for assembly of long DNA fragments.
The second general method for gene synthesis utilizes polymerase to fill in single-stranded gaps in the annealed pairs of oligonucleotides. After the polymerase reaction, single-stranded regions of oligonucleotides become double-stranded, and after digestion with restriction endonuclease, can be cloned directly or used for further assembly of longer sequences by ligating different double-stranded fragments. This approach is relatively independent of the secondary structure of oligonucleotides; however, after the polymerase reaction, each segment must be cloned. The cloning step significantly delays the synthesis of long DNA fragments and greatly decreases the efficiency of the approach. Additionally, this approach can be used for only relatively small DNA fragments and requires restriction endonuclease recognition sites to be introduced into the sequence.
Thus, the major essential disadvantages of existing approaches for the synthesis of DNA is low efficacy and the requirement that synthesized DNA must be amplified by cloning procedures, or by the PCR, before use. The main problem with existing approaches is that the long polynucleotide must be assembled from relatively short oligonucleotides utilizing either inefficient chemical or enzymatic synthesis. The use of short oligonucleotides for the synthesis of long polynucleotides can cause many problems due to multiple interactions of complementary bases, as well as problems related to adverse secondary structure of oligonucleotides. These problems lower the efficiency and widespread use of existing synthetic approaches.
Therefore, there exists a great need for an efficient means to make synthetic DNA of any desired sequence. Such a method could be universally applied. For example, the method could be used to efficiently make an array of DNA having specific substitutions in a known sequence which are expressed and screened for improved function. The present invention satisfies these needs by providing an efficient and powerful method for the synthesis of DNA. The method is generally referred to as the Exchangeable Template Reaction (ETR).